Micro RNAs (miRNA) and small interfering RNAs (siRNAs) are considered key regulators of posttranslational mRNA modifications, capable of affecting the expression of multiple genes simultaneously . Small RNAs have been identified in human testis, and hold promise for identifying major breakthroughs in the understanding of human reproduction. Comparative analysis of sequencing of miRNA data (human testis) showed that 70 % of human miRNA is highly conserved between species, thus allowing for relatively easy study of the mechanism of action of candidate miRNA in animal models. The current proposal aims to use multiplexed deep sequencing to identify new small RNAs, and to employ qRT-PCR to identify miRNAs critical in male infertility. Cell isolation will be used to enrich the population of different cells, allowing increased sensitivity to detect specific miRNAs in spermatogonia, spermatocytes, Leydig cells, and Sertoli cells. Expression profiles from fertile male testes, men with Sertoli cell only syndrome and maturation arrest will be evaluated. Expression of sperm miRNA in normal men and men with severe oligospermia (density <1 mil/ml) will be analyzed to determine if the ejaculated sperm miRNA profiling may be useful in diagnosis of genetic reasons for male infertility. Subsequent experiments will focus on a subset of conserved and X-linked miRNA interacting with set of genes important in reproduction. TruSeq mRNA profiles obtained from fertile and infertile patients will be used to experimentaly identify critical miRNA:mRNA targets. Selected targets will be confirmed further in transgenic models as part of U54 center collaborative effort. At the end ofthe proposed funding period, this work will identify and confirm a set of approximately 20-30 miRNA:mRNA interactions that are critical in male infertility, verify their target specificity in vitro, delineate their mechanisms of action, and assign them to known regulatory pathways. This will lay a solid groundwork for the development of in vivo models. These models can then be used to test whether delivery of exogenous miRNAs, or their inhibitors, can rescue the infertile phenotype. This work will also provide insights for the development of RNA-based therapies for infertility and other testicular disorders.